Packaging cells

ABSTRACT

The invention described herein allows the production of recombinant retroviruses (retroviral vector particles) from producer cells which are safer and of higher titer than normal. In addition, methods are provided for making helper cells which, when a recombinant retrovirus genome is introduced to make a producer line, produce particles that are targeted toward particular cell types. Methods are also provided for making recombinant retrovirus systems adapted to infect a particular cell type, such as a tumor, by binding the retrovirus or recombinant retrovirus in the particular cell type. Methods are also provided for producing recombinant retroviruses which integrate in a specific small number of places in the host genome, and for producing recombinant retroviruses from transgenic animals.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.07/586,603, which is a continuation-in-part of U.S. Ser. No. 07/565,606,filed Aug. 10, 1990, which is a continuation-in-part of U.S. Ser. No.07/395,932, filed Aug. 18, 1989, which is a continuation-in-part of U.S.Ser. No. 07/170,515, filed Mar. 21, 1988, which application is nowabandoned.

TECHNICAL FIELD

[0002] The present invention relates generally to retroviruses, and morespecifically, to recombinant retroviruses which are capable ofdelivering vector constructs to susceptible target cells. These vectorconstructs are typically designed to express desired proteins in targetcells, including proteins which can have a therapeutic effect in anumber of ways, and hence, constitute a “drug” transport system forallowing transport of proteins (or RNA) into cells. The specificity ofproteins (and RNA) for enzymatic reaction, for binding of cellularcomponents, for immunological action, or for other biological effects,allows for correspondingly specific actions on target cells if theprotein or RNA molecule can be transported into the cells. Such actionsinclude the repair of genetic defects, production of antisense RNA toblock cellular process, the enzymatic potentiation of prodrugs, andstimulation of the cellular immune system, as well as many othertherapies based on the intracellular production of proteins.

BACKGROUND OF THE INVENTION

[0003] Retroviruses are RNA viruses which can replicate and integrateinto a host cell's genome through a DNA intermediate. This DNAintermediate, or provirus, may be stably integrated into the host'scellular DNA. Due to their efficiency at integrating into host cells,retroviruses are considered to be one of the most promising vectors foruse in human gene therapy. These vectors have a number of propertiesthat lead them to be considered as one of the most promising techniquesfor genetic therapy of disease. These include: (1) efficient entry ofgenetic material (the vector genome) into cells; (2) an active efficientprocess of entry into the target cell nucleus; (3) relatively highlevels of gene expression; (4) minimal pathological effects on targetcells; and (5) the potential to target to particular cellular subtypesthrough control of the vector-target cell binding and thetissue-specific control of gene expression. For example, a foreign geneof interest may be incorporated into the retrovirus in place of thenormal retroviral RNA. When the retrovirus injects its RNA into a cell,the foreign gene is also introduced into the cell, and may then beintegrated into the host's cellular DNA as if it were the retrovirusitself. Expression of this foreign gene within the host results inexpression of the foreign protein by the host cell.

[0004] Most retroviruses which have been developed for gene therapy aremurine retroviruses. Briefly, these retroviruses exist in two forms, asproviruses integrated into a host's cellular DNA, or as free virions.The virion form of the virus contains the structural and enzymaticproteins of the retrovirus (including reverse transcriptase), two RNAcopies of the viral genome, and portions of the cell's plasma membranein which is embedded the viral envelope glycoprotein. The genome isorganized into four main regions: the Long Terminal Repeat (LTR), andthe qag, pol. and env genes may be found at both ends of the proviralgenome, is a composite of the 5′ and 3′ ends of the RNA genome, andcontains cis-acting elements necessary for the initiation andtermination of transcription. The three genes gag, pol, and env arelocated between the terminal LTRs. The gag and pol genes encode,respectively, internal viral structures and enzymatic proteins. The envgene encodes the envelope glycoprotein which confers infectivity andhost range specificity of the virus.

[0005] An important consideration in using retroviruses for gene therapyis the availability of “safe” retroviruses. Packaging cell lines havebeen developed to meet this concern. Briefly, this methodology employsthe use of two components, a retroviral vector and a packaging cell. Theretroviral vector contains long terminal repeats (LTRs), the foreign DNAto be transferred and a packaging sequence (i). This retroviral vectorwill not reproduce by itself because the genes which encode structuraland envelope proteins are not included within the vector. The packagingcell contains genes encoding the gag, pol, and env proteins, but doesnot contain the packaging signal “ψ” Thus, a packaging cell can onlyform empty virion particles by itself. Within this general method, theretroviral vector is introduced into the packaging cell, therebycreating a “producer cell.” This producer cell manufactures virionparticles containing only the retroviral vector's (foreign) DNA, andtherefore has previously been considered to be a safe retrovirus fortherapeutic use.

[0006] There are several shortcomings in the current use of thisapproach. One issue involves the generation of “live virus” (i.e.,competent replicating retrovirus) by the producer cell line.Preparations of human therapeutics which are contaminated withretroviruses are not currently considered suitable for use in humantherapy. For example, extreme measures are taken to exclude retroviralcontamination of for imaging and therapy. Live virus can in conventionalproducer cells when: (1) The vector genome and the helper genomesrecombine with each other; (2) The vector genome or helper genomerecombines with homologous cryptic endogenous retroviral elements in theproducer cell; or (3) Cryptic endogenous retroviral elements reactivate(e.g., xenotropic retroviruses in mouse cells).

[0007] Another issue is the propensity of mouse based producer lines topackage endogenous retroviral-vector-like elements (which can containonc gene sequences) at efficiencies close to that with which theypackage the desired vector. Such elements, because of their vector-likestructure, are transmitted to the target treatment cell at frequenciesthat parallel its transfer of the desired vector sequence.

[0008] A third issue is the ability to make sufficient vector particlesat a suitable concentration to: (1) treat a large number of cells (e.g.,10⁸-10¹⁰); and (2) manufacture vector particles at a commercially viablecost. Finally, the only producer lines currently used for transfer ofgenes to human cells are amphotropic producer lines, known for theeponymous murine retroviral envelope gene, which has receptors in mosthuman cells.

[0009] In order to construct safer packaging cell lines, researchershave generated additional deletions in the 3′ LTR and portions of the 5′LTR (see, Miller and Buttimore, Mol. Cell. Biol., 6:2895-2902, 1986).When such cells are used, two recombination events are necessary to formthe wild-type genome. Nevertheless, results from several laboratorieshave indicated that even when several mutations are present, wild-typevirus may still be generated (see, Bosselman et al., Mol. Cell. Biol.7:1797-1806, 1987; Danos and Mulligan, Proc. Nat'l. Acad. Sci. USA81:6460-6464, 1988).

[0010] Many of the helper cell lines that have been described to datehave been limited to a host cell range of murine, avian, rat and dogcell lines have been generated using amphotropic retroviral vectorsystems, which can infect human cells as well as a broad range of othermammalian cells (see, Sorge et al., Mol. Cell. Biol. 4:1720-1737, 1984),amphotropic packaging lines developed thus far have retained portions ofone or more of the viral LTRs, and, thus, even when multiple mutationsare present, have remained capable of generating a replication-competentgenome. Amphotropic vector systems with multiple mutations and reducedpropensities toward generating infectious virus generally exhibitunsatisfactorily low titres of retroviral particles.

[0011] One of the more recent approaches to constructing safer packagingcell lines involves the use of complementary portions of helper virus,divided among two separate plasmids, one containing gag and pol, and theother containing env (see, Markowitz et al., J. Virol. 62:1120-1124; andMarkowitz et al., Virology 167: 600-606, 1988. One benefit of thisdouble-plasmid system is that three recombination events are required togenerate a replication competent genome. Nonetheless, thesedouble-plasmid vectors have also suffered from the drawback of includingportions of the retroviral LTRs, and therefore remain capable ofproducing infectious virus. Cell lines containing both 3′ and 5′ LTRdeletions have been constructed, but have thus far not proven usefulsince they produce relatively low titers (Daugherty et al., J. Virol.63:3209-3212, 1989).

[0012] The present invention overcomes difficulties of prior packagingcell lines, and further provides other related advantages.

SUMMARY OF THE INVENTION

[0013] The present invention provides a method for producing recombinantretroviruses in which the retroviral genome is packaged in a capsid andenvelope, preferably through the use of a packaging cell. The packagingcells are provided with viral protein-coding sequences, preferably inthe form of two plasmids integrated into the genome of the cell, whichproduce all proteins necessary for production of viable retroviralparticles, a DNA viral construct which codes for an RNA which will carrythe desired gene, along with a packaging signal which will directpackaging of the RNA into the retroviral particles.

[0014] The present invention additionally provides a number oftechniques for producing recombinant retroviruses which can facilitate:

[0015] i) the production of higher titres from packaging cells;

[0016] ii) the production of higher titres of helper free recombinantretrovirus from packaging cell lines that are non-murine (to avoidproduction of recombinant or endogenously activated retroviruses, and toavoid packaging of defective murine retroviral sequences) and which willinfect human cells;

[0017] iii) the production of helper free recombinant retroviruses withhigher titres using alternative non-hybrid envelopes such as xenotropicor polytropic envelope proteins (to allow infection of cells poorlyinfectable with amphotropic recombinant retroviruses or to allowspecificity of cell type infection).

[0018] iv) packaging of vector constructs by means not involving the useof packaging cells;

[0019] v) the production of recombinant retroviruses which can betargeted for preselected cell lines;

[0020] vi) the construction of retroviral vectors with tissue-specific(e.g., tumor) promoters; and

[0021] vii) the integration of the proviral construct into a preselectedsite or sites in a cell's genome.

[0022] One technique for producing higher titres from packaging cellstakes advantage of the discovery that of the many factors which canlimit titre from a packaging cell, one of the most limiting is the levelof expression of the packaging proteins, namely, the gag, pol, and envproteins, as well as the level of expression of the retroviral vectorRNA from the proviral vector. This technique allows the selection ofpackaging cells which have higher levels of expression (i.e., producehigher concentrations) of the foregoing packaging proteins and vectorconstruct RNA. More specifically, this technique allows selection ofpackaging cells which produce high levels of what is referred to hereinas a “primary agent,” which is either a packaging protein (e.g., gag,pol, or env proteins) or a gene of interest to be carried into thegenome of target cells (typically as a vector construct). This isaccomplished by providing in packaging cells a genome carrying a gene(the “primary gene”) which expresses the primary agent in the packagingcells, along with a selectable gene, preferably downstream from theprimary gene. The selectable gene expresses a selectable protein in thepackaging cells, preferably one which conveys resistance to an otherwisecytotoxic drug. The cells are then exposed to a selecting agent,preferably the cytotoxic drug, which enables identification of thosecells which express the selectable protein at a critical level (i.e., inthe case of a cytotoxic drug, by killing those cells which do notproduce a level of resistance protein required for survival).

[0023] Preferably, in the technique briefly described above, theexpression of both the selectable and primary genes is controlled by thesame promoter. In this regard, it may be preferable to utilize a non-MLVretroviral 5′ LTR. In order to maximize titre of a recombinantretro-virus from packaging cells, this technique is first used to selectpackaging cells expressing high levels of all the required packagingproteins, and then is used to select which of these cells, followingtransfection with the desired proviral construct, produce the highesttitres of the recombinant retrovirus.

[0024] Techniques are also provided to select cells that produce highertitres of helper free recombinant retroviruses in non-murine cells.These cell lines produce recombinant retroviruses capable of efficientlyinfecting human cells. These techniques involve screening potentialparent cells for their ability to produce recombinant retroviruses inthe presence of a replicating virus. Subsequently, uninfected culturesof candidate cell lines chosen by the above procedure are infected witha vector expressing a retroviral gag/pol, and clones which synthesizehigh levels of gag/pol are identified. A clone of this type is thenreinfected with a vector expressing env, and clones expressing highlevel of env (and gag/pol) are identified. Within the context of thepresent invention, “high levels” means discernibly greater than thatseen in the standard mouse packaging line, PA317 on a Western blotanalysis. Many non-mouse cell lines such as human or dog have never beenknown to spontaneously generate competent retrovirus, do not carrypossible recombination partners for recombinant murine retroviralpackaging or gene sequences; and do not carry genes which make RNA whichmay be packaged by the MLV system. Techniques are provided to generatecell lines which produce high titres of recombinant retroviruses usingalternative envelopes such as xenotropic or polytropic by techniquessimilar to those described above. Such retroviruses may be used ininfecting amphotropic resistant cells (xenotropic envelope) or infectingonly a subset of cells (polytropic).

[0025] A technique suitable for producing recombinant retroviruses whichcan be targeted for preselected cell lines utilizes recombinantretroviruses having one or more of the following: an env gene comprisedof a cytoplasmic segment of a first retroviral phenotype, and anextracellular binding segment exogenous to the first retroviralphenotype (the binding segment being from a second viral phenotype orfrom another protein with desired binding properties which is selectedto be expressed as a peptide which will bind to the desired target);another viral envelope protein; another ligand molecule in place of thenormal envelope protein; or another ligand molecule along with anenvelope protein that does not lead to infection of the target celltype. Preferably, in the technique briefly described above, an env genecomprised of a cytoplasmic segment of a retroviral phenotype is combinedwith an exogenous gene encoding a protein having a receptor-bindingdomain to improve the ability of the recombinant retrovirus to bindspecifically to a targeted cell type, e.g., a tumor cell. In thisregard, it may be preferable to utilize a receptor-binding domain whichbinds to receptors expressed at high levels on the surface of the targetcell (e.g., growth factor receptors in tumor cells) or alternatively, areceptor-binding domain binding to receptors expressed at a relativelyhigher level in one tissue cell type (e.g., epithelial cells, ductalepithelial cells, etc., in breast cancer). One potential advantage totargeting with hybrid envelopes with specificity for growth factor oractivation receptors (like EGF or CD3 receptors is that binding of thevector itself may then lead to cell cycling, which is necessary forviral integration and expression. Within this technique, it may bepossible to improve and genetically alter recombinant retroviruses withspecificity for a given tumor by repeated passage of a replicatingrecombinant retrovirus in tumor cells; or by linking the vectorconstruct to a drug resistance gene and selecting for drug resistance.

[0026] Techniques for integrating a retroviral genome at a specific sitein the DNA of a target cell involve the use of homologous recombination,or alternatively, the use of a modified integrase enzyme which willrecognize a specific site on the target cell specific insertion allowsgenes to be inserted at sites on the target cells' DNA, which willminimize the chances of insertional mutagenesis, minimize interferencefrom other sequences on the DNA, and allow insertion of sequences atspecific target sites so as to reduce or eliminate the expression of anundesirable gene (such as a viral gene) in the DNA of the target cell.

[0027] It will be appreciated that any of the above-described techniquesmay be used independently of the others in particular situations, or canbe used in conjunction with one or more of the remainder of thetechniques.

[0028] These and other aspects of the present invention will becomeevident upon reference to the following detailed description andattached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1A depicts four plasmids designed to express retroviralproteins in mammalian cells. pSVgp and pRSVenv are cotransfected with aselectable marker, while pSVgp-DHFR and pRSVenv-phleo are the equivalentplasmids with the selectable marker placed downstream of the viralprotein-coding regions.

[0030]FIG. 1B depicts vectors which lead- to expression of: 1. MLV coreproteins (pSCV10); 2-4 MLV amphotropic env (pCMvenv AmDra, pCMVenvAnNhe, pMLPenv AmSph); 5. MLV xenotropic env (PCMV xeno). 6. MLV MCFenv(pCMV MCF); 7. MLV amphotropic env as a retroviral vector (pLARNL).

[0031]FIG. 1C depicts the results of the screening procedure forassessing the intrinsic ability of cell lines to make retroviral vectorsin the presence of helper virus (Example 2B).

[0032]FIG. 1D depicts the results of selecting clones of cells intowhich pSCV10 had been introduced and examining these clones for gagproduction as compared to PA317 by Western blots.

[0033]FIG. 1E depicts the results of Western blot experiments to comparelevels of amphotropic env in cell lysates from DA, CA, 2A and PA317.

[0034]FIG. 1F depicts the results of Western blot experiments to comparelevels of xenotropic env in cell lysates from transient transfections ofCF gag/pol and permanently expressing lines XF7, X6, X10 and PA317(ampho env).

[0035]FIG. 1G depicts the results of Western blot 10 experiments tocompare levels of MCF (polytropic) env in HT1080 derived clones, PA317(amphotropic env) and HX (xenotropic env).

[0036]FIG. 2 depicts three sites of fusion of HIV env and MOMLV envafter site-directed mutagenesis. The joint at the extracellular marginof the transmembrane region is designated as A, while B and C indicatelocations of joints at the middle of the transmembrane region andcytoplasmic margin, respectively. The numbering is according tonucleotide numbers (RNA Tumor Viruses, Vol. II, Cold Spring Harbor,1985). ST, SR, SE are the starts of tat, rev and env while TT, TR, andTE are the corresponding termination sites.

[0037]FIG. 3 depicts the substitution of U3 in a 5′ LTR by aheterologous promoter/enhancer in which can be fused to either the SacI, Bssh II or other site in the region.

[0038]FIG. 4 illustrates a representative method for crossing transgenicmice expressing viral protein or vector RNA. 30

DETAILED DESCRIPTION OF THE INVENTION

[0039] In one aspect, the present invention is based, in part, upon thediscovery of the major causes of low recombinant virus titres frompackaging cell lines (PCL), and of techniques to correct those causes.Basically, at least five factors may be postulated as causes for lowrecombinant virus titres:

[0040] 1. the limited availability of viral packaging proteins;

[0041] 2. the limited availability of retroviral vector RNA genomes;

[0042] 3. the limited availability of cell membrane for budding of therecombinant retroviruses;

[0043] 4. the limited intrinsic packaging efficiency of the retroviralvector genome; and

[0044] 5. the density of the receptor specific for the envelope of agiven retrovirus.

[0045] 6. The limited availability of host cell constituents (such asRNA or myristoylation, phosphorylation, glycosylation or proteolyticfunctions).

[0046] As noted above, the limited availability of viral packagingproteins is the initial limiting factor in recombinant retrovirusproduction from packaging cells. When the level of packaging protein inthe packaging cells is increased, titre increases to about 10⁵infectious units/milliliter, following which increasing packagingprotein level has no further effect on titres. However, titres can befurther augmented by also increasing the level of retroviral vectorgenome available for packaging. Thus, as described herein, it isadvantageous to select producer cells that manufacture the maximumlevels of packaging proteins and retroviral vector genomes. It has beendiscovered that the methods of identifying, and thus selecting,packaging cells and producer cells, described earlier under the sectionentitled “Background of the Invention,” tend to lead to selection ofmany producer cells which produce low titres for the reasons describedbelow.

[0047] The present invention takes advantage of the previouslydisadvantageous fact that the protein expression level of a genedownstream from the 5′ LTR or other promoter, and spaced therefrom by anintervening gene, is substantially less than if the intervening genewere absent. In the present invention, the selectable gene is placeddownstream from a gene of the packaging genome or the gene of interestcarried by the vector construct, but is still transcribed under thecontrol of the viral 5′ LTR or other promoter without any splice donoror splice acceptor sites. This accomplishes two things. First, since thepackaging genes or genes of interest are now upstream with nointervening gene between themselves and the promoter, theircorresponding proteins (packaging protein or protein of interest) willbe expressed at a higher level (five- to twentyfold) than the selectableprotein. Second, the selectable protein will be expressed on average ata lower level, with the distribution of level of expression shiftingtoward lower levels. However, the selection level for resistance tophleomycin remains the same, so that only the top-end expressing cellssurvive. The levels of the packaging protein or of the protein ofinterest will still be proportional, only in this case, a higher levelof selectable protein corresponds to a much higher level of packagingprotein or protein of interest.

[0048] Preferably, the foregoing procedure is performed using a plasmidcarrying one of the proviral gag/pol or env packaging genes, along witha first selectable gene. These cells are then screened for the cellsproducing the highest levels of protein by reaction with an antibodyagainst gag/pol (or possibly env), a second enzyme or labelled antibody,and then sorted on a fluorescence-activated cell sorter (FACS) ordetected on a western blot. Alternatively, other tests for protein levelmay be used. Subsequently, the procedure and screening are repeatedusing those selected cells, and the other of the gag/pol or envpackaging genes. In this step, a second selectable gene (different fromthe first) would be required downstream from the packaging gene and thecells producing the largest amount of the selected. This cell line is apackaging cell line (PCL) that may be used with any available vector.The procedure and screening are then repeated using the surviving cells,with a plasmid carrying the proviral vector construct bearing the geneof interest and a third selectable gene, different from the first orsecond selectable gene. As a result of this procedure, cells producinghigh titres of the desired recombinant retrovirus will be selected, andthese can be cultured as required to supply recombinant retrovirus. Inaddition, gag and pol can be independently introduced and selected.

[0049] Example 1 describes the construction of gag/pol and env plasmidsdesigned to use these procedures.

EXAMPLE 1 Plasmids Designed to Make High Levels of Packaging Proteins(FIG. 1)

[0050] 1. The 2.7 kb Xba I fragment from pPAM (Miller et al., Mol. Cell.Biol. 5:431, 1985), which contains the amphotrophic env segment, wascloned in pUC18 at the Xba I site, then removed with Hind III and Sma I.This fragment was cloned into the vector pRSV neo (Gorman et al., Mol.Cell. Biol. 2:1044, 1982; Southern et al., J. Mol. Appl. Genet. 1:327,1982) cut with Hind III and Pvu II, to give pRSV env. A 0.7 kb Bam HI toBstE II fragment from the plasmid pUT507 (Mulsant et al., Somat. Cell.Mol. Genet. 14:243, 1988) with the BstE II end filled in carries thephleo resistance coding sequence. The 4.2 kb Bam HI to Xho I fragment,the contiguous 1.6 kb Xho I to Xba I (Xba I filled in) from RSVenv, andthe phleo fragment were ligated to give pRSVenv-phleo.

[0051] 2. A fragment from the Pst I site at nucleotide 563 of MLV (RNATumor Viruses, Vol. II, Cold Spring Harbor, 1985) to the Sca I site at5870 was derived from pMLV-K (Miller et al., 1985, op. cit.) and clonedin the Pst I to Bam HI (Bam HI filled-in) fragment from p4aA8 (Jolly etal., Proc. Natl. Acad. Sci. USA 80:477, 1983) that has the SV40promoter, the pBR322 ampicillin resistance and origin of replication andthe SV40 poly A site. This gives pSVgp. pSVgpDHFR was made using thefollowing fragments: the 3.6 kb Hind III to Sal I fragment from pSVgpcontaining the SV40 promoter plus MLV gag and some pol sequences; the2.1 kb Sal I to Sca I fragment from PMLV-K with the rest of the polgene, the 3.2 kb Xba I (Xba I filled-in) to Pst I fragment from pF400with the DHFR gene plus poly A site, pBR322 origin and half theampicillin resistance gene; the 0.7 kb Pst I to Hind III fragment frompBR322 with the other half of the ampicillin resistance gene. This givespSVgp-DHFR. All these constructs are shown in FIG. 1. These plasmids canbe transfected into 3T3 cells or other cells, and high levels of gag,pol or env obtained.

[0052] An additional method for accomplishing selection is to use a geneselection in one round and its antisense in a subsequent round. Forexample, gag/pol may be introduced into an HPRT-deficient cell with theHPRT gene and selected for the presence of this gene using that mediawhich requires HPRT for the salvage of purines. In the next round, theantisense to HPRT could be delivered downstream to env and the cellselected in 6 thioguanine for the HPRT-deficient phenotype. Largeamounts of antisense HPRT would be required in order to inactivate theHPRT gene transcripts, assuming no reversion occurred. A further methodof accomplishing selection is described below. Co-transfection of a 10×stoichiometric excess of the expression vector over the separateselectable marker ensures high copy number of expression vector in drugresistant cell clones. In most of the examples noted herein, the gag/poland envelope expression vectors were introduced independently (i.e.,separate transfections) so that the two structural genes would notrecombine or concatamerize (as transfected integrated DNA tends to do),assuring that the genes are unlinked in the genome. The steady-statelevel of intracellular MLV gag/pol and env was measured by proteinimmunoblotting. The relative ease, sensitivity, and reproducibility ofimmunoblotting allowed rapid, quantitative analysis of a large number ofcell clones necessary to identify over-expressors of the MLV structuralproteins (gag/pol in particular) (see Example 2).

[0053] In addition to the gag/pol expressing constructs which begin atnucleotide 563 of MOMLV, several others can be constructed which containupstream lead sequences. It has been observed by Prats et al. (RNA TumorViruses Meeting, Cold Spring Harbor, N.Y., 1988) that a glycosylatedform of the gag protein initiates at nucleotide 357 and a translationenhancer maps in the region between nucleotides 200-270. Therefore,gag/pol expressing constructs may be made beginning at the Bal I site(nucleotide 212) or Eag I site (nucleotide 346) to include theseupstream elements and enhance vector production. A preferred method ofaccomplishing this is to include degenerate mutations to inactivate thepackaging signal found here, without affecting the coding potential ofthe nucleic acid.

[0054] Envelope Substitutions

[0055] The ability to express gag/pol and env function separately allowsfor manipulation of these functions independently. A cell line thatexpresses ample amounts of gag/pol can be used, for example, to addressquestions of titre with regard to env. One factor resulting in lowtitres is the density of appropriate receptor molecules on the targetcell or tissue. A second factor is the affinity of the receptor for theviral envelope protein. Given that env expression is from a separateunit, a variety of envelope genes (requiring different receptorproteins), such as xenotropic, polytropic, or amphotrophic envs from avariety of sources, can be tested for highest titres on a specifictarget tissue.

[0056] Envelope proteins from one retrovirus can often substitute, tovarying degrees, for that of another retrovirus. For instance, theenvelope of murine virus 4070A, HTLV I, GALV, and BLV can eachsubstitute for that of MOMLV, albeit with a lower efficiency (Cone andMulligan, Proc. Natl. Acad. Sci. USA 81:6349-53, 1984; Wilson et al., J.Virol. 63, 2374-78, 1989; Ban et al., J. Gen. Virol. 70:1987-93, 1989).To increase the number of cell types that could be infected withMLV-based vectors, PCLs were generated which individually express eitheramphotropic, xenotropic, or polytropic envelopes. Vector produced fromany of these PCLs can be used to infect any cell which contains thecorresponding distinct receptor (Rein and Schultz, Virology 136:144-52,1984). Some cell types may, for instance, lack the amphotropic receptorand thus be resistant to infection with amphotropic vector, but expressthe xenotropic receptor and therefore be infectable by xenotropicvector. One report suggests that xenotropic vector, in the presence ofreplication-complement xenotropic virus, may more effectively infecthuman hematopoietic progenitor cells (Eglitis et al., Biochem. Biophys.Res. Comm. 151:201-206, 1988). Xenotropic vector, in the presence ofreplication-competent xenotropic virus, also infects cells from otherspecies which are not easily infectable by amphotropic virus such asbovine, porcine, and equine (Delouis et al., Biochem. Biophys Res. Comm.169:80-14, 1990). The xenotropic PCLs will therefore be useful forveterinary purposes in these species. Another example would beutilization of the spleen focus-forming virus (SFFV) envelope gene whichmay allow targeting to cells containing the erythropoietin receptor (J.P. Li et al., Nature 343:762-764, 1990).

[0057] As a specific example, all of the amphotropic PCLs describedherein (canine and human fibroblasts) were infectable by xenotropicvector but were resistant to infection by amphotropic vector, presumablydue to the phenomenon of “viral interference” (cf. A. Rein, Virology120:251-57, 1982). The xenotropic PCL therefore allows the facileinfection of these amphotropic PCLs, which in turn produces10-100×-higher titre than PCLs whose vector has been introduced by othermeans (Miller et al., Somat. Cell Mol. Genet. 12:175-83, 1986). Inprinciple, a PCL expressing any viral envelope which can function withthe MLV vector and packaging system and whose corresponding cellularreceptor is found in a given PCL, is useful for allowing vectorinfection of that PCL.

[0058] Vector produced from the polytropic PCL described herein has amore restricted host range on human cells than vector produced fromeither amphotropic or xenotropic PCLs (see data below). The polytropicPCL may therefore be particularly useful for targeting vector to aspecific human cell type. The reduced homology between both xenotropicand polytropic envelopes with the MOMLV gag/pol and with the vectormakes these PCLs even less likely to generate replication-competentretrovirus by homologous recombination than amphotropic PCLs. Examplesof the use of these methods are set forth below (see Example 2).

[0059] Furthermore, envelopes from nonmurine retrovirus sources can beused for pseudotyping a vector. The exact rules for pseudotyping (i.e.,which envelope proteins will interact with the nascent vector particleat the cytoplasmic side of the cell membrane to give a viable viralparticle (Tato, Virology 88:71, 1978) and which will not (Vana, Nature336:36, 1988), are not well characterized. However, since a piece ofcell membrane buds off to form the viral envelope, molecules normally inthe membrane are carried along on the viral envelope. Thus, a number ofdifferent potential ligands can be put on the surface of viral vectorsby manipulating the cell line making gag and pol in which the vectorsare produced or choosing various types of cell lines with particularsurface markers. One type of surface marker that can be expressed inhelper cells and that can give a useful vector-cell interaction is thereceptor for another potentially pathogenic virus. The pathogenic virusdisplays on the infected cell surface its virally specific protein(e.g., env) that normally interacts with the cell surface marker orreceptor to give viral infection. This reverses the specificity of theinfection of the vector with respect to the potentially pathogenic virusby using the same viral protein-receptor interaction, but with thereceptors on the vector and the viral protein on the cell.

[0060] It may be desirable to include a gene which encodes for anirrelevant envelope protein which does not lead to infection of targetcells by the vector so produced, but does facilitate the formation ofinfectious viral particles. For example, one could use human Sup T1cells as a helper line. This human T-cell line expresses CD4 moleculesat high levels on its surface. Conversion of this into a helper line canbe achieved by expressing gag/pol with appropriate expression vectorsand also, if necessary, the Moloney ecotropic env gene product as anirrelevant (for human cells) envelope protein (the Moloney ecotropic envonly leads to infection of mouse cells). Vectors produced from such ahelper line would have CD4 molecules on their surfaces and therefore becapable of infecting only cells which express HIV env, such asHIV-infected cells.

[0061] In addition, hybrid envelopes (as described below) can be used inthis system as well, to tailor the tropism (and effectively increasetitres) of a retroviral vector. A cell line that expresses ample amountsof a given envelope gene can be employed to address questions of titrewith regard to gag and pol.

[0062] Furthermore, it is also possible to add ligand moleculesexogenously to the viral particles which either incorporate themselvesin the lipid envelope or can be linked chemically to the lipid orprotein constituents.

[0063] Cell Lines

[0064] The most common packaging cell lines used for MOMLV vectorsystems (psi2, PA12, PA317) are derived from murine cell lines. Thereare several reasons why a murine cell line is not the most suitable forproduction of human therapeutic vectors:

[0065] 1. They are known to contain endogenous retroviruses, some ofwhich are closely related in sequence and viral type to the MLV vectorsystem used here.

[0066] 2. They contain nonretroviral or defective retroviral sequencesthat are known to package efficiently.

[0067] 3. There may be deleterious effects caused by the presence ofmurine cell membrane components.

[0068] Several non-murine cell lines are potential parents for packaginglines. These include Vero cells which are used in Europe to preparepolio vaccine, WI38 which are used in the U.S. in vaccine production,CHO cells which are used in the U.S. for TPA preparation, D17 or otherdog cells that may have no endogenous viruses, and those described inExample 2.

[0069] The most important safety concern for the production ofretroviral vectors is the inherent propensity of retroviral PCLs togenerate replication-competent retrovirus after introduction of a vector(Munchau et al., Virology 176:262-65, 1990). This can occur in at leasttwo ways: 1) homologous recombination can occur between the therapeuticproviral DNA and the DNA encoding the MOMLV structural genes (“gag/pol”and “env”) present in the PCL (discussed below under “Generation ofPCLs”); and 2) generation of replication-competent virus by homologousrecombination of the proviral DNA with the very large number ofdefective endogenous proviruses found in murine cells (Steffen andWeinberg, Cell 15:1003-10, 1978); Canaani and Aaronson, Proc. Natl.Acad. Sci., USA 76:1677-81, 1979; Stoye and Coffin, J. Virol. 61:2659-691987). In addition, even murine cell lines lacking vector can producevirus spontaneously or after induction, (e.g., xenotropic virus whichcan replicate in human cells, Aaronson and Dunn, J. Virol. 13:181-85,1974; Stephenson and Aaronson, Proc. Natl. Acad. Sci., USA 71:4925-29,1974; Aaronson and Stephenson, Biochem. Biophys. Acta 458:323-54, 1976).Another safety concern with the utilization of murine cells for theproduction of murine retroviral vectors is the observation that some ofthe many endogenous proviral genes (retrovirus-like genes) in the murinegenome are expressed, recognized by the retroviral structural geneproducts of murine PCLs, and delivered and expressed in target cellswith an efficiency at least comparable to that of the desired vector(Scolnick et al., J. Virol. 29:964-72, 1979; Scadden et al., J. Virol.64:424-27, 1990). These observations strongly suggest that murine celllines are an unsafe choice for the production of murine retroviralvectors for human therapeutics. To circumvent the inherent safetyproblems associated with murine cells, PCLs have been generatedexclusively from non-murine cell lines (e.g., canine and human celllines) which are known to lack genomic sequences homologous to that ofMOMLV by hybridization analysis (data not shown) (Martin et al., Proc.Natl. Acad. Sci., USA 78:4892-96, 1981). Those skilled in the art willrecognize that the packaging cells described herein will have a low, butinherent capability of packaging random RNA molecules. Such RNAmolecules will not be permanently transmitted to the pseudo-infectedtarget cell.

[0070] In addition to issues of safety, the choice of host cell line forthe PCL is of importance because many of the physical (such asstability) and biological properties (such as titre) of retroviralparticles are dictated by the properties of the host cell. For instance,the host cell must efficiently express (transcribe) the vector RNAgenome, prime the vector for first strand synthesis with a cellulartRNA, tolerate and covalently modify the MLV structural proteins(proteolysis, glycosylation, myristylation, and phosphorylation), andthe maturing virion buds from the cell membrane, carrying many of themembrane components with it. For example, it has been found that vectormade from the mouse packaging line PA317 is retained by a 0.3 micronfilter, while that made from the CA line described herein will passthrough.

EXAMPLE 2 Packaging Cell Selection

[0071] A. MLV Structural Gene Expression Vectors

[0072] To decrease the possibility of replication-competent virus beinggenerated by genetic interactions between the MLV proviral vector DNAand the structural genes of the PCL, separate expression vectors, eachlacking the viral LTR, were generated to express the gag/pol and envgenes independently. To further decrease the possibility of homologousrecombination with MLV vectors and the resultant generation ofreplication-competent virus, minimal sequences other than the proteincoding sequences were used. In order to express high levels of the MLVstructural proteins in the host cells, strong transcriptional promoters(CMV early and Ad5 major late promoters) were utilized. An example ofthe construction of a MoMLV gag/pol expression vector (pSCV10, see FIG.1B.1) follows:

[0073] 1. The 0.7 Kb HinCII/XmaIII fragment encompassing the humancytomegalovirus (CMV) early transcriptional promoter (Boshart et al.,Cell 41:521-30, 1985) was isolated.

[0074] 2. A 5.3 Kb PstI(partial)/ScaI fragment from the MoMLV proviralplasmid, MLV-K (Miller et al., Mol. Cell Biol. 5:531, 1985) encompassingthe entire gag/pol coding region was isolated.

[0075] 3. A 0.35 Kb DraI fragment from SV40 DNA (residues 2717-2363)encompassing the SV40 late transcriptional termination signal wasisolated.

[0076] 4. Using linkers and other standard recombinant DNA techniques,the CMV promoter-MoMLV gag/pol-SV40 termination signal was ligated intothe bluescript vector SK⁺.

[0077] An example of the construction of an MLV amphotropic envelopeexpression vector (pCMVenvAmDra, see FIG. 1B.2) follows.

[0078] 1. A 2.7 Kb XbaI/NheI fragment containing the coding sequence ofamphotropic envelope from the 4070A proviral clone (Chattopadhyay etal., J. Virol. 39:777-91, 1981) was isolated.

[0079] 2. Using linkers and other standard DNA techniques, the CMV earlypromoter and SV40 late termination signal described for the gag/polexpression above (pSCV10) were ligated in the order CMVpromoter-envelope-termination signal.

[0080] A second example of the construction of an MLV amphotropicenvelope expression vector (PCMVenvAmNhe, see FIG. 1B.3) follows.

[0081] 1. A 2.7 Kb XbaI/NheI fragment containing the coding sequence ofamphotropic envelope from the 4070A proviral clone described above wasisolated.

[0082] 2. Using linkers and other standard recombinant DNA techniques,the CMV early promoter described for the gag/pol expression above(pSCV10) was ligated in the plasmid pUC18 in the order CMVpromoter-envelope (no added transcriptional termination signal).

[0083] A third example of the construction of an MLV amphotropicenvelope expression vector (pMLPenvAmSph, see FIG. 1B.4) follows.

[0084] 1. A 0.9 Kb EcoRI/HindIII fragment containing the Adenovirus 5left end, major late transcriptional promoter, and tripartite leadersequence was isolated.

[0085] 2. A 0.85 Kb EcoRI/BamHI fragment containing the SV40 small tintron and transcriptional termination signal from clone pJD204 (De Witet al., Mol. Cell. Biol. 7:725-37, 1987) was isolated.

[0086] 3. A 3 Kb SphI/SmaI fragment containing the coding sequence ofamphotropic envelope from the 4070A proviral clone described above wasisolated.

[0087] 4. Using linkers and other standard recombinant DNA techniques,the MLP, amphotropic envelope and the SV40 termination signal wereligated in plasmid pBR322 in the order MLP-envelope-SV40.

[0088] An example of the construction of an MLV xenotropic envelopeexpression vector (pCMMVxeno, see FIG. 1B.5) follows.

[0089] 1. A 2.2 Kb NaeI/NheI fragment containing the coding region ofthe xenotropic envelope obtained from clone NZB9-1 (O'Neill et al., J.Virol. 53:100-106, 1985) was isolated.

[0090] 2. Using linkers and other standard recombinant DNA techniques,the CMV early promoter and SV40 late termination signal described forthe gag/pol expression above (pSCV10) were ligated in the order CMVpromoter-envelope-termination signal.

[0091] An example of the construction of an MLV polytropic envelopeexpression vector (pCMVMCF, see FIG. 1B.6) follows.

[0092] 1. A 2 Kb BamHI/NheI fragment containing the coding region of thepolytropic envelope obtained from clone MCF-247W (Holland et al., J.Virol. 53:152-57, 1985) was isolated.

[0093] 2. Using linkers and other standard recombinant DNA techniques,the CMV early promoter and SV40 late termination signal described forthe gag/pol expression above (pSCV10) were ligated in the order CMVpromoter-envelope-termination signal.

[0094] An example of the construction of an MLV ampho env Neo⁺retroviral vector (pLARNL, FIG. 1B.7) follows.

[0095] 1. The vector PLRNL vector (Emi et al., J. Virol. 65:1202-1207,1991) was digested with BamHI.

[0096] 2. A 2.7 Kb XbaI fragment containing the envelope protein codingregion of retrovirus 4070A (Chattopadhyay et al., J. Virol. 39:777-91,1981) was isolated.

[0097] 3. Fragments from procedures 1 and 2 above were ligated.

[0098] B. Host Cell Selection

[0099] Host cell lines were screened for their ability to efficiently(high titre) rescue a drug resistance retroviral vector (A alpha N2)using replication competent retrovirus to produce the gag/pol and envstructural genes (“MA” virus). Titre was measured from confluentmonolayers 16 h after a medium change by adding filtered supernatants(0.45 um filters) to 5×10⁴ NIH 3T3 TK⁻ cells on a 6 cm tissue cultureplate in the presence of 4 μg/ml polybrene followed by selection inG418.

[0100] Data from the screening process is shown in FIG. 2. Among thenon-murine cell lines which demonstrate the ability to packageMoMLV-based vector with high titre are the cell lines CF2, D17, 293, andHT1080. These cell lines were used herein as examples, although anyother cells may be tested by such means.

[0101] C. Generation of Packaging Cell

[0102] (i) gag/pol Intermediate

[0103] As examples of the generation of gag/pol intermediates for PCLproduction, D17, 293, and HT1080 were co-transfected with 1 ug of themethotrexate resistance vector, pFR400 (Graham and van der Eb, Virology52:456-67, 1973), and 10 ug of the MOMLV gag/pol expression vector,pSCV10 (above) by calcium phosphate co-precipitation (D17 and HT1080,see Graham and van der Eb, Virology 52:456-67, 1973), or lipofection(293, see Felgner et al., Proc. Natl. Acad. Sci., USA 84:7413-17, 1987).After selection for transfected cells in the presence of the drugsdipyrimidol and methotrexate, individual drug resistant cell colonieswere expanded and analyzed for MOMLV gag/pol expression by extracellularreverse transcriptase (RT) activity (modified from Goff et al., J.Virol. 38:239-48, 1981) and intracellular p30^(gag) by western blotusing anti p30 antibodies (goat antiserum #77S000087 from the NationalCancer Institute). This method identified individual cell clones in eachcell type which expressed 10-50× higher levels of both proteins comparedwith that of a standard mouse amphotropic PCL, PA317 (FIG. 1D and Table1). TABLE 1 PROPERTIES OF MoMLV GAG/POL-EXPRESSING CELLS LARNL RTp30^(gag) TITRE CELL NAME ACTIVITY (CPM) EXPRESSION (CFU/ML) 3T3 800 −N.D. PA317 1350 +/− 1.2 × 10³ D17 800 − N.D. D17 4-15 5000 + + + + + 1.2× 10⁴ D17 9-20 2000 + + + 6.0 × 10³ D17 9-9 2200 + + 1.0 × 10³ D17 9-166100 + + + + + 1.5 × 10⁴ D17 8-7 4000 − N.D. HT1080 900 − N.D. HTSCV2116400 + + + + + 8.2 × 10³ HTSCV25 7900 + + + 2.8 × 10³ HTSCV42 11600 + +8.0 × 10² HTSCV26 4000 − <10 293 600 − N.D. 293 2-3 6500 + + + + +   7 ×10⁴ 293 5-2 7600 + + + + + N.D.

[0104] The biological activity of these proteins was tested byintroducing a retroviral vector, LARNL (see FIG. 1B) which expressesboth the amphotropic envelope and a Neo⁺ marker which confers resistanceto the drug, G418. In every case, co-expression of gag/pol in the cellline and env from the vector allowed efficient packaging of the vectoras determined by cell-free transfer of G418 resistance to 3T3 cells(titre). Titre was measured from confluent monolayers 16 h after amedium change by adding filtered supernatants (0.45 um filters) to 5×10⁴NIH3T3 TK⁻ cells on a 6 cm tissue culture plate in the presence of 4ug/ml polybrene followed by selection in G418. Significantly, the vectortitres from the cell lines correlated with the levels of p30^(gag)(Table 1). Since the level of env should be the same in each clone andis comparable to the level found in PA317 (data not shown), thisindicates that titre was limited by the lower levels of gag/pol in thesecells (including PA317). The titre correlated more closely with thelevels of p30^(gag) than with the levels of RT.

[0105] (ii) Conversion of gag/pol Lines Into Amphotropic Packaging Lines

[0106] As examples of the generation of amphotropic PCLs, the gag/polover-expressors for 293 (termed 2-3) and D17 (termed 4-15) wereco-transfected by the same techniques described above except that 1 ugof the phleomycin resistance vector, pUT507 (Mulsant et al., Somat. CellMol. Genet. 14:243-52, 1988), and 10 ug of the amphotropic envelopeexpression vectors, pMLPenvAmSph (for 2-3) or pCMVenvAmNhe (for 4-15)were used. After selection for transfected cells in the presence ofphleomycin, individual drug resistant cell colonies were expanded andanalyzed for intracellular gp80^(env) expression by western blot usinganti gp7o (goat antiserum #79S000771 from N.C.I.). Several clones wereidentified which expressed relatively high levels of both gag/pol andampho env (PCLs, see FIG. 1 for representative data).

[0107] In another example of the generation of an ampho PCL, CF2 cellswere electroporated (cf. Chu et al., Nucl. Acids Res. 15:1311-26, 1987)with 2 ug of the phleomycin resistance marker, pUT507, 10 ug of pSCV10(above), and 10 ug of pCMVenvAmNhe (above). After selection fortransfected cells in the presence of phleomycin, individual drugresistant cell colonies were expanded and analyzed for intracellularexpression of MLV p30^(gag) and gp80^(env) proteins by western blotusing specific antisera. A clone was identified which expressedrelatively high levels of both gag/pol and ampho env (FIG. 1E).

[0108] (iii) Performance of Amphotropic Packaging Cell Lines

[0109] A number of these ampho PCLs were tested for their capacity topackage retroviral vectors by measuring titre after the introduction ofretroviral vectors (Table 2). The measurements were performed usinguncloned PCLs, so that the average performance of the lines wascalculated. TABLE 2 VECTOR TITRE AND HELPER VIRUS GENERATION INAMPHOTROPIC PCLs VECTOR TITRE^(a) (+/− HELPER VIRUS^(b)) CELL TYPE b-GalKT-1 N2 PA317 3.5 × 10² (N.D.) 1.0 × 10⁴ (N.D.) 3.0 × 10⁵ (+)^(c) CA 5.0× 10⁴ (N.D.) 3.0 × 10⁵ (−)^(d) 2.0 × 10⁶ (−)^(d) 2A 4.0 × 10⁴ (N.D.) 2.0× 10⁵ (−)^(e) N.D. DA N.D. N.D. 2.0 × 10⁵ (−)^(d) DA2 N.D. 3.9 × 10⁵(−)^(d) N.D.

[0110] Highest titres are obtained when retroviral vectors wereintroduced into PCLs by infection (Miller et al., Somat. Cell Mol.Genet. 12:175-83, 1986). However, although amphotropic MLV vectors areknown to infect these host cell types, the PCLs are blocked forinfection by ampho vector since they express ampho env (“viralinterference”). To overcome this problem, vectors containing other viralenvelopes (such as xenotropic env or VSV G protein, which bind to cellreceptors other than the ampho receptor) were generated in the followingmanner. Ten ug of the vector DNA of interest was co-transfected with 10ug of DNA which expresses either xeno env (pCXvxeno, above) or a VSV Gprotein expression vector, MLP G, onto a cell line which expresses highlevels of MOMLV gag/pol such as 2-3 cell (see above). The resultantvector containing xenotropic env or VSV G protein, respectively, wasproduced transiently in the co-transfected cells and after 2 days cellfree supernatants were added to the potential PCLs, and vector-infectedcells were identified by selection in G418. Both types of vectorefficiently infected the ampho-blocked cells and after G418 selectioncell free supernatants were collected from the confluent monolayers andtitred on NIH 3T3 TK⁻ cells as described above. The cell clones with thehighest titre were chosen as PCLs and referred to as DA (D17 ampho), 2A(293 ampho), and CA (CF2 ampho), respectively. In no case was helpervirus detected in the currently described PCLs, even when a retroviralvector (N2) which has a high probability of generating helper virus(Armentano et al., J. Virol. 62:1647-50, 1987) was introduced into thePCLs and the cells passaged for as long as 2 months (3 months for vectorKT-3). On the other hand, the same vector introduced into the PA317 cellline generated helper virus within 3 weeks of continual passaging.

[0111] (iv) Conversion of gag/pol Lines into Xenotropic Packaging CellLines

[0112] As examples of the generation of xenotropic PCLS, the gag/polover-expressors for D17 (4-15) and HT1080 (SCV21) were co-transfected bythe same techniques described above except that 1 ug of either thephleomycin resistance vector, pUT507 (for SCV21), or the hygromycin Bresistance marker, pY3 (for 4-15, see Blochlinger and Diggelmann, Mol.Cell Biol. 4:2929-31, 1984), and 10 ug of the xenotropic envelopeexpression vector, pCMVxeno (above) was used. After selection fortransfected cells in the presence of phleomycin or hygromycin,respectively, individual drug resistant cell colonies were expanded andanalyzed for intracellular expression of MLV p30^(gag) and gp75^(env)proteins by western blot using specific antisera. Clones were identifiedwhich expressed relatively high levels of both gag/pol and xeno env(FIG. 1F).

[0113] (v) Performance of Xenotropic Packaging Cell Lines

[0114] A number of these potential xeno PCLs were tested for theircapacity to package retroviral vectors by measuring titre after theintroduction of retroviral vectors (Table 3). TABLE 3 VECTOR TITRE ONXENOTROPIC PCLs KT-1 TITRE (CFU/ML) CELL CLONE ON HT1080 CELLS HT1080SCV21 XF1 1.0 × 10⁵ XF7 1.0 × 10⁵ XF12 (HX) 4.5 × 10⁵ D17 4-15 X6 9.0 ×10⁴ X10 (DX) 1.3 × 10⁵ X23 8.0 × 10⁴

[0115] As described above, vector containing VSV G protein was producedtransiently in 2-3 cells. After 2 days, cell free supernatants wereadded to the xeno PCLs and after G418 selection cell free supernatantswere collected from the confluent monolayers and titred as describedabove except that HT1080 cells, which are infectable by xeno vector, wasused instead of NIH 3T3 TK⁻ cells which are resistant to xeno vector.The cell clones with the highest titre were chosen as PCLs and referredto as DX (D17 xeno) and HX (HT1080 xeno), respectively.

[0116] The propensity of the PCLs described above to generate helpervirus was tested even more stringently by co-cultivating ampho and xenoPCLs containing the vector, N2. Since ampho vector can infect the xenoPCLs and vice versa, this allows continuous cross-infection events, eachof which increases the probability of generating helper virus. As anexample, 2A cells containing N2 were co-cultivated with HX cellscontaining N2. After 23 days, the cultures were still free of ampho andxeno viruses as judged by a vector rescue assay on 293 or Mus dunnicells, both of which can detect ampho and xeno viruses (Table 4). TABLE4 HIGH STRINGENCY ANALYSIS FOR PCL TENDENCY TO GENERATE HELPER VIRUSTEST MATERIAL HELPER VIRUS ASSAY AMPHOTROPIC VIRUS + XENOTROPIC VIRUS +PA317 + N2 (21d) + 2A + HX + N2 (23d) −

[0117] (vi) Conversion of gag/pol Lines into Polytropic Packaging CellLines

[0118] As an example of the generation of a polytropic PCL, the gag/polover-expressor for HT1080 (SCV21) was co-transfected by the sametechniques described above, except that 1 ug of the phleomycinresistance vector, pUT507, and 10 ug of the polytropic envelopeexpression vector, pCMVMCF (above) was used. After selection fortransfected cells in the presence of phleomycin, individual drugresistant cell colonies were expanded and analyzed for intracellularexpression of MLV gp₇₀env protein by western blot using specificantiserum. Clones were identified which expressed relatively high levelsof both gag/pol (not shown) and polytropic env (FIG. 1G).

[0119] (vii) Performance of Polytropic Packaging Cell Lines

[0120] One of these potential poly PCLs (clone 3) was tested for thecapacity to package retroviral vectors by measuring titre after theintroduction of retroviral vectors (Table 5). TABLE 5 HOST-RANGE OFPOLYTROPIC VECTOR FROM HP CELLS CELL LINE SPECIES β-Gal TITRE 3T3 MURINE1.0 × 10⁴ PA317 MURINE 1.0 × 10⁴ 208F/C5 RAT 4.0 × 10⁴ Mv-1-Lu MINK 5.0× 10³ FRhL MACAQUE <10 HT1080 HUMAN <10 HeLa HUMAN <10 WI 38 HUMAN <10DETROIT 551 HUMAN <10 SUP TI HUMAN <10 CEM HUMAN <10 U937 HUMAN <10 293HUMAN 2.0 × 10⁴ AAT HUMAN <10 Vandenberg HUMAN <10

[0121] This cell clone was chosen as PCL and referred to as HP (HT1080poly). As described above, vector containing VSV G protein was producedtransiently in 2-3 cells and after 26 days, cell free supernatants wereadded to the polytropic PCL (HP). After G418 selection, cell freesupernatants were collected from the confluent monolayers and titred asdescribed above on a variety of cell lines. The infection of human cellswas very restricted, with all cell lines tested being negative with theexception of 293 cells.

[0122] Although the factors that lead to efficient infection of specificcell types by retroviral vectors are not completely understood, it isclear that because of their relatively high mutation rate, retrovirusesmay be adapted for markedly improved growth in cell types in whichinitial growth is poor, simply by continual reinfection and growth ofthe virus in that cell type (the adapter cell). This can also beachieved using viral vectors that encode some viral functions (e.g.,env), and which are passed continuously in cells of a particular typewhich have been engineered to have the functions necessary to complementthose of the vector to give out infectious vector particles (e.g.,gag/pol). For example, one can adapt the murine amphotropic virus 4070Ato human T-cells or monocytes by continuous growth and reinfection ofeither primary cell cultures or permanent cell lines such as Sup T1(T-cells) or U937 (monocytes). Once maximal growth has been achieved, asmeasured by reverse transcriptase levels or other assays of virusproduction, the virus is cloned out by any of a number of standardmethods, the clone is checked for activity (i.e., the ability to givethe same maximal growth characteristic on transfection into the adaptercell type) and this genome used to make defective helper genomes and/orvectors which in turn, in an appropriately manufactured helper orproducer line, will lead to production of viral vector particles whichinfect and express in the adapter cell type with high efficiency(10⁷-10⁹ infectious units/ml).

[0123] VII. Alternative Viral Vector Packaging Techniques

[0124] Two additional alternative systems can be used to producerecombinant retroviruses carrying the vector construct. Each of thesesystems takes advantage of the fact that the insect virus, baculovirus,and the mammalian viruses, vaccinia and adenovirus, have been adaptedrecently to make large amounts of any given protein for which the genehas been cloned. For example, see Smith et al. (Mol. Cell. Biol. 3:12,1983); Piccini et al. (Meth. Enzymology, 153:545, 1987); and Mansour etal. (Proc. Natl. Acad. Sci. USA 82:1359, 1985).

[0125] These viral vectors can be used to produce proteins in tissueculture cells by insertion of appropriate genes into the viral vectorand, hence, could be adapted to make retroviral vector particles.

[0126] Adenovirus vectors are derived from nuclear replicating virusesand can be defective. Genes can be inserted into vectors and used toexpress proteins in mammalian cells either by in vitro construction(Ballay et al., EMBO J. 4:3861, 1985) or by recombination in cells(Thummel et al., J. Mol. Appl. Genetics 1:435, 1982).

[0127] One preferred method is to construct plasmids using theadenovirus Major Late Promoter (MLP) driving: (1) gag/pol, (2) env, (3)a modified viral vector construct. A modified viral vector construct ispossible because the U3 region of the 5′ LTR, which contains the viralvector promoter, can be replaced by other promoter sequences (see, forexample, Hartman, Nucl. Acids Res. 16:9345, 1988). This portion will bereplaced after one round of reverse transcriptase by the U3 from the 3′LTR.

[0128] These plasmids can then be used to make adenovirus genomes invitro (Ballay et al., op. cit.), and these transfected in 293 cells (ahuman cell line making adenovirus E1A protein), for which the adenoviralvectors are defective, to yield pure stocks of gag/pol, env andretroviral vector carried separately in defective adenovirus vectors.Since the titres of such vectors are typically 10⁷-10¹¹/ml, these stockscan be used to infect tissue culture cells simultaneously at highmultiplicity. The cells will then be programmed to produce retroviralproteins and retroviral vector genomes at high levels. Since theadenovirus vectors are defective, no large amounts of direct cell lysiswill occur and retroviral vectors can be harvested from the cellsupernatants.

[0129] Other viral vectors such as those derived from unrelatedretroviral vectors (e.g., RSV, MMTV or HIV) can be used in the samemanner to generate vectors from primary cells. In one embodiment, theseadenoviral vectors are used in conjunction with primary cells, givingrise to retroviral vector preparations from primary cells.

[0130] In some cases, gene products from other viruses may be used toimprove the properties of retroviral packaging systems. For instance,HIV rev protein might be included to prevent splicing of HIV env or HIVgag/pol MLV vectors or HIV sor might increase the infectivity of T cellsby free virus as it does with HIV (See Fischer et al., Science237:888-893, 1987).

[0131] In an alternative system (which is more truly extracellular), thefollowing components are used:

[0132] 1. gag/pol and env proteins made in the baculovirus system in asimilar manner as described in Smith et al. (supra) (or in other proteinproduction systems, such as yeast or E. coli);

[0133] 2. viral vector RNA made in the known T7 or SP6 or other in vitroRNA-generating system (see, for example, Flamant and Sorge, J. Virol.62:1827, 1988);

[0134] 3. tRNA made as in (2) or purified from yeast or mammalian tissueculture cells;

[0135] 4. liposomes (with embedded env protein); and

[0136] 5. cell extract or purified necessary components (whenidentified) (typically from mouse cells) to provide env processing, andany or other necessary cell-derived functions.

[0137] Within this procedure (1), (2) and (3) are mixed, and then envprotein, cell extract and pre-liposome mix (lipid in a suitable solvent)added. It may, however, be necessary to earlier embed the env protein inthe liposomes prior to adding the resulting liposome-embedded env to themixture of (1), (2), and (3). The mix is treated (e.g., by sonication,temperature manipulation, or rotary dialysis) to allow encapsidation ofthe nascent viral particles with lipid plus embedded env protein in amanner similar to that for liposome encapsidation of pharmaceuticals, asdescribed in Gould-Fogerite et al., Anal. Biochem. 148:15, 1985). Thisprocedure allows the production of high titres of replicationincompetent recombinant retroviruses without contamination withpathogenic retroviruses or replication-competent retroviruses.

[0138] VIII. Cell Line-Specific Retroviruses—“Hybrid Envelope”

[0139] The host cell range specificity of a retrovirus is determined inpart by the env gene products. For example, Coffin, J. (RNA TumorViruses 2:25-27, Cold Spring Harbor, 1985) notes that the extracellularcomponent of the proteins from murine leukemia virus (MLV) and RousSarcoma virus (RSV) are responsible for specific receptor binding. Thecytoplasmic domain of envelope proteins, on the other hand, areunderstood to play a role in virion formation. While pseudotyping (i.e.,the encapsidation of viral RNA from one species by viral proteins ofanother species) does occur at a low frequency, the envelope protein hassome specificity for virion formation of a given retrovirus. The presentinvention recognizes that by creating a hybrid env gene product (i.e.,specifically, an env protein having cytoplasmic regions and exogenousbinding regions which are not in the same protein molecule in nature)the host range specificity may be changed independently from thecytoplasmic function. Thus, recombinant retroviruses can be producedwhich will specifically bind to preselected target cells.

[0140] In order to make a hybrid protein in which the receptor bindingcomponent and the cytoplasmic component are from different retroviruses,a preferred location for recombination is within the membrane-spanningregion of the cytoplasmic component. Example 10 describes theconstruction of a hybrid env gene which expresses a protein with the CD4binding portion of the HIV envelope protein coupled to the cytoplasmicdomain of the MLV envelope protein.

EXAMPLE 3 Hybrid HIV-MLV Envelopes

[0141] A hybrid envelope gene is prepared using in vitro mutagenesis(Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492, 1985) to introduce a newrestriction site at an appropriate point of junction. Alternatively, ifthe two envelope sequences are on the same plasmid, they can be joineddirectly at any desired point using in vitro mutagenesis. The end resultin either case is a hybrid gene containing the 5′ end of the HIV gp 160and the 3′ end of MLV pl5E. The hybrid protein expressed by theresulting recombinant gene is illustrated in FIG. 2 and contains the HIVgp120 (CD4 receptor binding protein), the extracellular portion of HIVgp 41 (the gp 120 binding and fusigenic regions), and the cytoplasmicportion of MLV piSE, with the joint occurring at any of several pointswithin the host membrane. A hybrid with a fusion joint at thecytoplasmic surface (joint C in FIG. 2) causes syncytia when expressedin Sup T1 cells. The number of apparent syncytia are approximatelyone-fifth that of the nonhybrid HIV envelope gene in the same expressionvector. Syncytia with the hybrid occurs only when the rev protein isco-expressed in trans. A hybrid with a fusion joint at the extracellularsurface (joint A in FIG. 2) gives no syncytia while hybrid B (in themiddle of transmembrane regions) gives approximately five-fold lesssyncytium on Sup T1 cells than hybrid C.

[0142] While Example 3 illustrates one hybrid protein produced from twodifferent retroviruses, the possibilities are not limited toretroviruses or other viruses. For example, the receptor binding portionof human interleukin-2 may be combined with the envelope protein of MLVto target vectors to cells with IL-2 receptors. In this case, arecombination would preferably be located in the gp 70 portion of theMLV env gene, leaving an intact p15E protein. Furthermore, the foregoingtechnique may be used to create a recombinant retrovirus with anenvelope protein which recognizes antibody Fc segments. Monoclonalantibodies which recognize only preselected target cells only could thenbe bound to such a recombinant retrovirus exhibiting such envelopeproteins so that the retrovirus would bind to and infect only thosepreselected target cells. Alternatively, a hybrid envelope with thebinding domain of avidin would be useful for targeting cells' “images”in a patient or animal with biotinylated antibodies or other ligands.The patient would first be flooded with antibodies, and then antibodybinding nonspecifically allowed to clear from the patient's system,before administering the vector. The high affinity (10⁻¹⁵) of the avidinbinding site for biotin would then allow accurate and efficienttargeting to the original tissue identified by the monoclonal “image.”

[0143] The approach may also be used to achieve tumor-specific targetingand killing by taking advantage of three levels of retroviral vectorspecificity; namely, cell entry, gene expression, and choice of proteinexpressed. Retroviral vectors enter cells and exert their effects atintracellular sites. In this respect their action is quite unique. Usingthis property, and the three levels of natural retroviral specificity(above), retroviral vectors may be engineered to target and kill tumorcells.

[0144] The overall goal of targeting of retrovirus to tumor cells may beaccomplished by two major experimental routes; namely, a) selection intissue culture (or in animals) for retrovituses that grow preferentiallyin tumor cells; or b) construction of retroviral vectors with tissue(tumor)-specific promoters with improvements being made by in vitropassage, and negative and positive drug-sensitivity selection.

[0145] Vectors suitable for selectively infecting selected cell types,such as a tumor cell, may generally be prepared by (a) continuouslypassaging a virus in cells of the selected cell type until the virus hasgenetically mutated and a predominant fast growing strain has evolved;(b) isolating the mutated and fast growing strain; (c) identifying andisolating the components of the mutated strain responsible for thepreferential growth of the mutated virus; (d) inserting the identifiedand isolated components as substitutes for counterpart components in aproducer cell based upon the virus (prior to continuous passage); and(e) culturing the producer cell to produce the vector.

[0146] At least four selective protocols may be utilized to select forretrovirus which grow preferentially in tumor cells; namely, 1) “EnvSelection by Passage In Vitro,” wherein selection of retrovirus withimproved replicative growth ability is accomplished by repeated passagein tumor cells; 2) “Selection with a Drug Resistance Gene,” whereingenetic selection for tumor “specific” retroviruses is based on viralconstructs containing a linked drug resistance gene; 3) “Hybrid-Env,”wherein selection (by protocol #1 or #2, above) of retrovirus withtumor-“specificity” is initiated from a construct containing a hybridenvelope gene which is a fusion of a tumor receptor gene (i.e., ananti-tumor antibody H-chain V-region gene fused with env; or, a growthreceptor fused with env); in this case selection begins at a favorablestarting point, e.g., an env which has some specificity for tumor cells;or 4) “Selection by Passage In Vitro and Counter Selection byCo-cultivation with Normal Cells,” wherein growth in tumor cells isselected-for by repeated passage in mixtures of drug-resistant tumorcells and drug-sensitive normal cells.

[0147] With respect to retroviral vector constructs carrying tissue(tumor)-specific promoters, biochemical markers with different levels oftissue-specificity are well known, and genetic control throughtissue-specific promoters is understood in some systems. There are anumber of genes whose transcriptional promoter elements are relativelyactive in rapidly growing cells (i.e., transferring receptor, thymidinekinase, etc.) and others whose promoter/enhancer elements are tissuespecific (i.e., HBV enhancer for liver, PSA promoter for prostate).Retroviral vectors and tissue-specific promoters (present either as aninternal promoter or within the LTR) which can drive the expression ofselectable markers and cell cycle genes (i.e., drug sensitivity, Ecogpt; or HSVTK in TK-cells). Expression of these genes can be selectedfor in media containing mycophenolic acid or HAT, respectively. In thismanner, tumor cells containing integrated provirus which activelyexpresses the drug resistance gene will survive. Selection in thissystem may involve selection for both tissue-specific promoters andviral LTRs. Alternatively, specific expression in tumor cells, and notin normal cells, can be counter-selected by periodically passaging virusonto normal cells, and selecting against virus that express Eco gpt orHSVtk (drug sensitivity) in those cells (by thioxanthine or acyclovir).Infected cells containing integrated provirus which express Eco gpt ortk phenotype will die and thus virus in that cell type will be selectedagainst.

[0148] IX. Site-Specific Integration

[0149] Targeting a retroviral vector to a predetermined locus on achromosome increases the benefits of gene-delivery systems. A measure ofsafety is gained by direct integration to a “safe” spot on a chromosome,i.e., one that is proven to have no deleterious effects from theinsertion of a vector. Another potential benefit is the ability todirect a gene to an “open” region of a chromosome, where its expressionwould be maximized. Two techniques for integrating retroviruses atspecific sites are described below.

[0150] (ii) Integrase Modification

[0151] Another technique for integrating a vector construct intospecific, preselected sites of a target cell's genome involves integrasemodification.

[0152] The retrovirus pol gene product is generally processed into fourparts: (i) a protease which processes the viral gag and pol products;(ii) the reverse transcriptase; and (iii) RNase H, which degrades RNA ofan RNA/DNA duplex; and (iv) the endonuclease or “integrase.”

[0153] The general integrase structure has been analyzed by Johnson etal. (Proc. Natl. Acad. Sci. USA 83:7648-7652, 1986). It has beenproposed that this protein has a zinc binding finger with which itinteracts with the host DNA before integrating the retroviral sequences.

[0154] In other proteins, such “fingers” allow the protein to bind toDNA at particular sequences. One illustrative example is the steroidreceptors. In this case, one can make the estrogen receptor, respondingto estrogens, have the effect of a glucocorticoid receptor, respondingto glucocorticoids, simply by substituting the glucocorticoid receptor“finger” (i.e., DNA binding segment) in place of the estrogen receptorfinger segment in the estrogen receptor gene. In this example, theposition in the genome to which the proteins are targeted has beenchanged. Such directing sequences can also be substituted into theintegrase gene in place of the present zinc finger. For instance, thesegment coding for the DNA binding region of the human estrogen receptorgene may be substituted in place of the DNA binding region of theintegrase in a packaging genome. Initially, specific integration wouldbe tested by means of an in vitro integration system (Brown et al., Cell29:347-356, 1987). To confirm that the specificity would be seen invivo, this packaging genome is used to make infectious vector particles,and infection of and integration into estrogen-sensitive andestrogen-nonsensitive cells compared in culture.

[0155] Through use of this technique, incoming viral vectors may bedirected to integrate into preselected sites on the target cell'sgenome, dictated by the genome-binding properties of site-specificDNA-binding protein-segments spliced into the integrase genome. It willbe understood by those skilled in the art that the integration sitemust, in fact, be receptive to the fingers of the modified integrase.For example, most cells are sensitive to glucocorticoids and hence theirchromatin has sites for glucocorticoid receptors. Thus, for most cells,a modified integrase having a glucocorticoid receptor finger would besuitable to integrate the proviral vector construct at thoseglucocorticoid receptor-binding sites.

[0156] X. Production of Recombinant Retroviral Vectors in TransgenicAnimals

[0157] Two problems previously described with helper line generation ofretroviral vectors are: (a) difficulty in generating large quantities ofvectors; and (b) the current need to use permanent instead of primarycells to make vectors. These problems can be overcome with producer orpackaging lines that are generated in transgenic animals. These animalswould carry the packaging genomes and retroviral vector genomes. Currenttechnology does not allow the generation of packaging cell lines anddesired vector-producing lines in primary cells due to their limitedlife span. The current technology is such that extensivecharacterization is necessary, which eliminates the use of primary cellsbecause of senescence. However, individual Lines of transgenic animalscan be generated by the methods provided herein which produce thepackaging functions, such as gag, pol or env. These lines of animals arethen characterized for expression in either the whole animal or targetedtissue through the selective use of housekeeping or tissue-specificpromoters to transcribe the packaging functions. The vector to bedelivered is also inserted into a line of transgenic animals with atissue-specific or housekeeping promoter. As discussed above, the vectorcan be driven off such a promoter substituting for the U3 region of the5′ LTR (FIG. 3). This transgene could be inducible or ubiquitous in itsexpression. This vector, however, is not packaged. These lines ofanimals are then mated to the gag/pol/env animal and subsequent progenyproduce packaged vector. The progeny, which are essentially identical,are characterized and offer an unlimited source of primary producingcells. Alternatively, primary cells making gag/pol and env and derivedfrom transgenic animals can be infected or transfected in bulk withretrovirus vectors to make a primary cell producer line. Many differenttransgenic animals or insects could produce these vectors, such as mice,rats, chickens, swine, rabbits, cows, sheep, fish and flies. The vectorand packaging genomes would be tailored for species infectionspecificity and tissue-specific expression through the use oftissue-specific promoters and different envelope proteins. An example ofsuch a procedure is illustrated in FIG. 4.

[0158] Although the following examples of transgenic production ofprimary packaging lines are described only for mice, these procedurescan be extended to other species by those skilled in the art. Thesetransgenic animals may be produced by microinjection or gene transfertechniques. Given the homology to MLV sequences in mice genome, thefinal preferred animals would not be mice.

EXAMPLE 4 Production of Gag/Pol Proteins Using Housekeeping Promotersfor Ubiquitous Expression in Transgenic Animals

[0159] An example of a well-characterized housekeeping promoter is theHPRT promoter. HPRT is a purine salvage enzyme which expresses in alltissues. (See Patel et al., Mol. Cell Biol. 6:393-403, 1986 and Meltonet al., Proc. Natl. Acad. Sci. 81:2147-2151, 1984). This promoter isinserted in front of various gag/pol fragments (e.g., Bal I/Sca I; AatII/Sca I; Pst I/Sca I of MOMLV; see RNA Tumor Viruses 2, Cold SpringHarbor Laboratory, 1985) that are cloned in Bluescript plasmids(Strategene, Inc.) using recombinant DNA techniques (see Maniatis etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982).The resulting plasmids are purified (Maniatis et al., op. cit.) and therelevant genetic information isolated using Geneclean (Bio 101) orelectroelution (see Hogan et al. (eds.), Manipulating the Mouse Embryo:A Laboratory Manual, Cold Spring Harbor, 1986).

[0160] These fully characterized DNAs are microinjected in thepronucleus of fertilized mouse ova at a concentration of 2 ug/ml.Live-born mice are screened by tail blot analyses (see Hogan et al., op.cit.). Transgenic-positive animals are characterized for expressionlevels of gag/pol proteins by immunoprecipitation of radiolabeledprimary cells, such as fibroblast (see Harlow et al. (eds.), Antibodies:A Laboratory Manual, Cold Spring Harbor, 1988). Animals then bred tohomozygosity for establishment of animal lines that producecharacterized levels of gag/pol.

EXAMPLE 5 Production of env Proteins/Hybrid Envelope Proteins UsingHousekeeping Promoters for Ubiquitous Expression in Transgenic Animals

[0161] This example utilizes the HPRT promoter for expression of eitherenvelope or hybrid envelope proteins. The envelope proteins can be fromany retrovirus that is capable of complementing the relevant gag/pol, inthis case that of MLV. Examples are ecotropic MLV, amphotrophic MLV,xenotropic MLV, polytropic MLV, or hybrid envelopes. As above, theenvelope gene is cloned behind the HPRT promoter using recombinant DNAtechniques (see Maniatis et al., op. cit.). The resulting “minigene” isisolated (see Hogan et al., op. cit.), and expression of envelopeprotein is determined (Harlow et al., op. cit.). The transgenic envelopeanimals are bred to homozygosity to establish a well-characterizedenvelope animal.

EXAMPLE 6 Production of gag/pol-env Animals Using Housekeeping Promotersfor Ubiquitous Expression in Transgenic Animals

[0162] This uses the well-characterized gag/pol animals, as well as theanimals for the establishment of a permanent gag/pol/envelope animalline. This involves breeding to homozygosity and the establishment of awell-characterized line. These lines are then used to establish primarymouse embryo lines that can be used for packaging vectors in tissueculture. Furthermore, animals containing the retroviral vector are bredinto this line.

EXAMPLE 7 Production of Tissue-Specific Expression of gag/pol-env orHybrid Envelope in Transgenic Animals

[0163] This example illustrates high level expression of the gag/pol,envelope, or hybrid envelope in specific tissues, such as T-cells. Thisinvolves the use of CD2 sequences (see Lang et al., EMBO J. 7:1675-1682,1988) that give position and copy number independence. The 1.5 kb BamHI/Hind III fragment from the CD2 gene is inserted in front of gag/pol,envelope, or hybrid envelope fragments using recombinant DNA techniques.These genes are inserted into fertilized mouse ova by microinjection.Transgenic animals are characterized as before. Expression in T-cells isestablished, and animals are bred to homozygosity to establishwell-characterized lines of transgenic animals. Gag/pol animals aremated to envelope animals to establish gag/pol-env animals expressingonly in T-cells. The T-cells of these animals are then a source forT-cells capable of packaging retroviral vectors. Again, vector animalscan be bred into these gag/pol-env animals to establish T-cellsexpressing the vector.

[0164] This technique allows the use of other tissue-specific promoters,such as milk-specific (whey), pancreatic (insulin or elastase), orneuronal (myelin basic protein) promoters. Through the use of promoters,such as milk-specific promoters, recombinant retroviruses may beisolated directly from the biological fluid of the progeny.

EXAMPLE 8 Production of Either Housekeeping or Tissue-SpecificRetroviral Vectors in Transgenic Animals

[0165] The insertion of retroviruses or retroviral vectors into the germline of transgenic animals results in little or no expression. Thiseffect, described by Jaenisch (see Jahner et al., Nature 298:623-628,1982), is attributed to methylation of 5′ retroviral LTR sequences. Thistechnique would overcome the methylation effect by substituting either ahousekeeping or tissue-specific promoter to express the retroviralvector/retrovirus. The U3 region of the 5′ LTR, which contains theenhancer elements, is replaced with regulatory sequences fromhousekeeping or tissue-specific promoters (see FIG. 20). The 3′ LTR isfully retained, as it contains sequences necessary for polyadenylationof the viral RNA and integration. As the result of unique properties ofretroviral replication, the U3 region of the 5′ LTR of the integratedprovirus is generated by the U3 region of the 3′ LTR of the infectingvirus. Hence, the 3′ is necessary, while the 5′ U3 is dispensable.Substitution of the 5′ LTR U3 sequences with promoters and insertioninto the germ line of transgenic animals results in lines of animalscapable of producing retroviral vector transcripts. These animals wouldthen be mated to gag/pol-env animals to generate retroviral-producinganimals (see FIG. 4).

[0166] From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method of selecting packaging cells which produce high levels of aprimary agent selected from a packaging protein and a gene product ofinterest, comprising: (a) providing in packaging cells a genomecomprising a primary gene which expresses a primary agent therein, and aselectable gene which expresses a selectable protein therein at lowerlevels than the primary agent, the expression levels of the primary geneand selectable gene being proportional; (b) exposing the packaging cellsto a selecting agent which enables identification of those cells whichexpress the selectable protein at a critical level; and (c) detectingthose packaging cells which express high levels of the primary agent. 2.A method of producing a recombinant retrovirus, comprising: generatinggag, pol, and env proteins from a cell line infected by a recombinantvirus which is capable of producing the proteins; and contacting theproteins with viral vector RNA, tRNA, liposomes, and a cell extract tocomplement missing functions for particle assembly, so as to producerecombinant retroviruses carrying the viral vector RNA.
 3. A method ofproducing a recombinant retrovirus, comprising: (a) generatingrecombinant viral vectors which separately or in combination, code forgag/pol, env and a retroviral vector genome; (b) producing high titrestocks of the vectors; and (c) co-infecting primary or other cells togenerate recombinant retroviral vectors.
 4. A method of producing arecombinant retrovirus, comprising growing a producer cell having agenome comprising: (a) a gene of interest along with a packaging signalof a first retroviral phenotype; (b) gag and pol genes of the firstretroviral phenotype, absent a packaging signal; (c) a hybrid env geneabsent a packaging signal, the product of said hybrid env genecomprising a cytoplasmic segment of the first retroviral phenotype, anda binding segment exogenous to the first retroviral phenotype.
 5. Amethod of producing a recombinant retrovirus, comprising growing aproducer cell having a genome comprising: (a) a gene of interest alongwith a packaging signal of a first retroviral phenotype; (b) gag and polgenes of the first retroviral phenotype, absent a packaging signal; and(c) a gene coding for a ligand which is expressed on the surface of theproducer cell and which is subsequently exhibited on the surface of thevector particle.
 6. The method of claim 5 wherein the ligand is CD4. 7.A hybrid env gene useful for preparing a retrovirus which canselectively carry a gene of interest to a target cell, the env genecoding for: (a) a cytoplasmic segment of a first retroviral phenotype;and (b) a binding segment exogenous to the first retroviral phenotype,the binding segment being capable of selectively binding to the targetcell.
 8. A method of producing a recombinant retrovirus which is capableof integrating its genome into a preselected site on a target cell'sgenome, comprising: packaging a vector in a capsid and envelope, andincluding in the viral particle a modified form of integrase which iscapable of integrating the retroviral genome into the preselected site.9. A method of producing recombinant retroviruses, comprising: mating atransgenic animal or insect containing a gag/pol-env viral construct,with a transgenic animal or insect containing a vector constructcontaining a promoter; isolating the progeny of said transgenic animalsor insects; isolating selected cells from the progeny; growing saidcells in an appropriate medium; and isolating recombinant retrovirusesfrom the cells.
 10. A method of producing a transgenic packaging animalor insect, comprising: mating a transgenic animal or insect containing avector construct coding for some, but not all viral proteins necessaryfor packaging, with a transgenic animal or insect containing a vectorconstruct coding for the remainder of said necessary viral proteins; andisolating the progeny of said transgenic animals or insects.
 11. Themethod of claim 10, further comprising the step of mating said progenywith a transgenic animal or insect containing a vector construct, toproduce primary cells capable of producing high titre recombinantretrovirus.
 12. The method of claim 10, further comprising the step ofinfecting cells explanted from said progeny with a recombinantretrovirus containing a vector construct to produce primary cellscapable of producing high titre recombinant retrovirus.
 13. A non-mousepackaging cell line that produces at least a ten-fold increase in viralpackaging protein, as compared to a standard mouse amphotropic packagingcell line.
 14. The cell line of claim 13 wherein the viral packagingprotein is the gag/pol protein.
 15. The cell line of claim 13 whereinthe cell line is an amphotropic packaging cell line.
 16. The packagingcell line of any one of claims 13-15 wherein the packaging cell line,upon introduction of a vector construct, produces at least a ten-foldincrease in vector titre as compared to a standard mouse amphotropicpackaging cell line.
 17. The packaging cell line of any one of claims13-15 wherein the packaging cell line, upon introduction of a vectorconstruct, produces vector particles capable of infecting human cells.18. A xenotropic packaging cell line which, upon introduction of avector construct, is capable of producing vector particles substantiallyuncontaminated by replication competent virus.
 19. The packaging cellline of claim 18 wherein the cell line produces at least equal vectortitre as compared to a standard mouse amphotropic packaging cell linewhen HT1080 cells are infected.
 20. A polytropic packaging cell line.21. The packaging cell line of claim 20 which, upon introduction of avector construct, is capable of producing vector particles substantiallyuncontaminated by replication competent virus.
 22. The packaging cellline of claim 20 wherein the packaging cell line, upon introduction of avector construct, produces at least a ten-fold increase in vector titreas compared to a standard mouse amphotropic packaging cell line when 293cells are infected.
 23. A polytropic packaging cell line wherein thepackaging cell line, upon introduction of a vector construct, producesvector particles capable of infecting cells of kidney lineage, but notcells of fibroblast, epithelial, T-cell or monocyte lineage.
 24. Anon-mouse packaging cell line carrying on separate operons the genes forgag/pol and env, said operons lacking retroviral LTR sequences andwhich, upon introduction of an N2 type vector construct, producessubstantially no helper virus after at least twenty days passage inculture.
 25. The cell line of claim 24 wherein the cell line is anamphotropic packaging cell line.
 26. The cell line of claim 24 whereinthe cell line is a polytropic packaging cell line.
 27. The cell line ofclaim 24 wherein the cell line is a xenotropic packaging cell line. 28.A method of producing a recombinant retrovirus, comprising: (a)introducing packaging genes from a retroviral vector system into a cellline, said cell line having substantially no endogenous proviruses whichproduce transcripts packageable by the retroviral vector system; and (b)selecting for cells that produce at least a tenfold increase in viralpackaging protein as compared to a standard mouse amphotropic packagingcell line, and that, upon introduction of a vector construct, produce atleast a ten-fold increase in vector titre as compared to a standardmouse amphotropic packaging cell line.
 29. A method of producing arecombinant retrovirus, comprising: (a) introducing packaging genes froma retroviral vector system capable of infecting human cells into a cellline, said cell line having substantially no endogenous proviruses whichproduce transcripts packageable by the retroviral vector system; and (b)selecting for cells that, upon introduction of a vector construct,produce vector titres at least equivalent to those of a standard mouseamphotropic packaging cell line, and which produce vector particlescapable of infecting human cells.
 30. A cell line selected from thegroup consisting of CA, 2A, DA, DA2, DX, HX, and HP.
 31. A method ofproducing a vector capable of infecting a selected cell type,comprising: (a) continuously passaging a virus in cells of the selectedcell type until the virus has genetically mutated and a predominant fastgrowing strain has evolved; (b) isolating the mutated and fast growingstrain; (c) identifying and isolating the components of the mutatedstrain responsible for the preferential growth of the mutated virus; (d)inserting the identified and isolated components as substitutes forcounterpart components in a producer cell based upon the virus prior toits continuous passage; and (e) culturing the producer cell to producethe vector.